This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. Recent studies have focused on: 1) analysis of the solution conformation and dynamics of the ribonuclease H (RNase H) domain of HIV reverse transcriptase, and the characterization of substrate-induced active site formation; 2) understanding the protein-mediated interactions of RT substrates and inhibitors; 3) understanding the complex structural maturation process that converts the initial p66 protein into the p66/p66' homodimer, and finally the p66/p51 heterodimer; 4) understanding the basis for the subunit-specific elimination of one RH domain 5) identification of potential targets for development of drugs that would interfere with the structural maturation process. During the past year, this effort has become more dominant as we have worked to identify ligands that can interfere with the metamorphic transition that is essential for the structuralmaturation of the enzyme. In addition, we evaluate other potential targets that may be both interesting and amenable to the research tools, primarily NMR spectroscopy, in which the group has expertise. A recent convergence of interests has led to studies of the role that the protein CYREN, cell cycle regulator of non-homologous end joining, that has been shown to be involved in modulating enzymes involved in the final integration step in which viral DNA is inserted into the host chromosomal DNA. Project 1. Targeting RT structural maturation. HIV-1 RT is an essential enzyme that supports the viral life cycle and is currently targeted with two classes of clinically approved drugs: nucleoside RT inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI). The mechanism of NRTI inhibition is transparent, as these drugs become phosphorylated and compete with the dNTP substrates, generally being incorporated at the primer terminus. Alternatively, the NNRTI inhibitors bind to a pocket that is not present in the unliganded enzyme. This remarkable observation apparently indicates that the structure of the apo-enzyme is dynamic, with the binding pocket appearing only intermittently. During the past year, we have evaluated ligands that can interfere with the structural maturation of the p66 monomer into the mature, active p66/p51 RT heterodimer. These studies have built on our previous determination of the monomer structure, on the identification of ligand binding sites that have been reported by other groups, and on the use of in silico modeling studies to evaluate other ligands that can potentially target these sites. Although we have not identified any high affinity ligands, we have identified a number of lower affinity ligands that interact with different sites on the monomer and show some ability to interfere with the metamorphic transition that is essential for dimer formation. It is anticipated that this work can form the starting point for further development of higher affinity ligands that target the RT structural maturation process. Project 2. Interaction of the modulator of retrovirus infection (MRI) with Ku80. The protein MRI has previously been identified as an endogenous cell protein that participates in the non-homologous end joining (NHEJ) pathway for repair of DNA double strand breaks. Further, there is evidence that MRI plays a critical role in the process whereby the proviral DNA is integrated into the chromosome of the host cell. These activities are thought to be dependent on interactions with the Ku70/Ku80 heterodimer. During the past year, we have characterized the interaction of an N-terminal Ku binding motif (KBM) derived from MRI with an N-terminal Ku80 domain, the von Willebrand factor A-like domain (vWA domain). A crystal structure corresponding to the interaction of this MRI-derived Ku binding motif with the isolated Ku80 vWA domain derived from the frog which shares 60% sequence identity with the human protein has been determined. Additional interactions of MRI are currently being explored.